Adult neurons are crucial and precious cell types controlling human body functions. Neuron injury following stroke and other neurologic diseases causes a significant loss of function. My long-term goal is to investigate the molecular mechanisms of neuronal injury and develop novel therapeutic strategies to treat patients suffering neurologic dysfunction. Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in glutamate neurotoxicity and cerebral infarction, which is a primary cause of subsequent morbidity and mortality following stroke. PARP-1 knockout mice are robustly resistant to stroke. Thus, it is critically important to understand the molecular mechanisms underlying PARP-1-dependent cell death (parthanatos) in stroke. Mitochondrial apoptosis-inducing factor (AIF) release and translocation to the nucleus causes chromatinolysis, which is the commitment point for parthanatos. However, little is known how AIF induces chromatinolysis and neuronal death after its nuclear translocation, since AIF itself has no endonuclease activity. Through an unbiased 17K human protein chip screening and a second siRNA-based PARP-1-dependent cell viability high-throughput screening, thirteen hits including AIF interactor 18 and protein arginine methyltransferase 6 (PRMT6) were identified. Here we focus on AIF interactor 18 as our preliminary data showed that knockdown of AIF interactor 18 is as protective as PARP-1 knockdown in parthanatos and it possesses hitherto unidentified endonuclease activity. Therefore, we name AIF interactor 18 as PAAN1 (Parthanatos-dependent AIF-Associated Nuclease 1). In the mentored K99 phase, we will first define the role of PAAN1 in ischemic neuronal death both in vitro and in vivo. We will then determine the specific PAAN1 endonuclease activity and determine if its endonuclease activity is required for neuronal injury. Histone post-translational modifications may regulate PAAN1 recognition in tightly wrapped genomic DNA to induce chromatinolysis and cell death after PARP-1 activation. PRMT6, a histone methyltransferase, was identified to be involved in parthanatos. In the independent R00 phase, I will study the role of PRMT6 in PAAN1-mediated ischemic cell death to understand the mechanism of PAAN1 recruitment to the DNA damage sites. I will further identify and characterize the functional PAAN1-interacting proteins required for parthanatos using two high-throughput screens, in order to understand how PAAN1 endonuclease activity is regulated by its networks. The successful completion of this project will yield important insights into cellular control of PAAN1 endonuclease activity and parthanatos and may provide new targets for developing innovative therapeutic approaches to treat patients with stroke. The mentored approach will be conducted in the laboratories of Drs. Valina and Ted Dawson and Dr. Raymond Koehler at the Johns Hopkins University School of Medicine. I have access to the resources available to Drs. Dawson and Koehler laboratories and the Neuroregeneration and Stem Cell Programs in Institute for Cell Engineering. To successfully accomplish the proposed research, during the mentored phase, I intend to take epigenetics courses and acquire skills in experimental stroke models and bioinformatics technologies, as well as grant writing and teaching skills. This training will allow me to expand my expertise and help my transition into a successful independent academic researcher.